Abstract

Background: Titanium (Ti) and Ti alloys are known to have good mechanical properties with high corrosion resistance and excellent biocompatibility. Doping of silver ions onto a titanium dioxide based surface resulted in the movement of the absorption to a longer wavelength due to a change in the electronic and optical properties of TiO2 and thereby increasing the antibacterial effect.

Aim: This work was done to investigate the effectivity of nanoparticles of TiO2 doped with 0.3% wt of silver prepared by sol-gel technique, as an anti-bacterial agent against Pseudomonas aeruginosa from clinical samples.

Materials and Methods: TiO2 with 0.3% silver as additive was systematically assessed on 25 Pseudomonas aeruginosa isolates from various clinical samples and incubated in dark, in visible light and under UV radiation for the antibacterial effect. After each hour of incubation, the isolates were inoculated onto Mueller Hinton agar.

Results: The viable colonies diminished within 2 h of incubation under UV irradiation, while under visible light, the number of viable colonies was highly reduced after 3 h. Under dark, the reduction was considerably slower, taking 6 h, while in all the light conditions the control, which had no exposure to nanoparticles, showed no reduction in the number of colonies.

Conclusion: TiO2 with Ag as an additive is highly effective as an antibacterial agent on many human pathogens including Pseudomonas aeruginosa, which is notorious in causing hospital acquired infections. TiO2 with 0.3 wt (%) Ag is most effective under UV irradiation and least in dark.

Keywords

Introduction

Microbial infections, especially nosocomial infections are one
of the most notorious causes of morbidity and mortality in the
health industries. The patient suffering from a hospital
acquired infection has a longer hospital stay and as a result,
there is an increase in health care costs [1]. Lowering of the
immune status of the patients especially due to a surgical
intervention further enhances the chances of an infection.

Pseudomonas aeruginosa is a gram negative, non-sporing,
non-capsulated, straight or slightly curved rod shaped
bacterium, which may occur singly, in pairs or sometimes in
short chains [2]. They are intrinsically highly resistant
organisms known to occur even in disinfectants, respiratory
equipment, common areas such as on taps, mops, sinks etc. It
is also one of the notorious organisms that are capable of
forming biofilms leading to a variety of hospital acquired
infections [3].

Infections due to P. aeruginosa normally evolve to a pattern of
chronic and persistent infections. The organisms undergo
phenotypic changes frequently characterized by the production
of a polysaccharide called alginate [4]. This phenotype called
mucoid phenotype is associated with a difficulty in eradicating
the organism, thereby eliciting a major inflammatory response
and poor prognosis [4-6].

Titanium (Ti) and Ti alloys are known to have good
mechanical properties with high corrosion resistance and
excellent biocompatibility and are commonly found in
orthopaedic prostheses, orthodontics, joint replacement and so
on [7-10]. Although titanium products are seen very often in
the dental appliances, its use in biomedical appliances has not
been well studied.

Silver (Ag) is a non- specific, noble metal nanoparticle whose
antibacterial effect has been well documented and is known to
be a broad spectrum anti-bactericidal as well as a fungicidal
[11]. It has a good stability in the environment as a result difficult for the microorganisms to develop resistance against it
[12-14]. There is documentation of the therapeutic window of
Ag to be small and larger doses are required for a cytotoxic
effect [15,16].

It has been proposed that the photoactivity of Ago/Ag2O
deposited onto TiO2 lead to the photoexcitation of Ag2O rather
than Ago and acts as active sites which are responsible for the
enhancement of the photocatalytic effect. Ago is said to
contribute to the stability if the molecule [17]. These silver
ions are known to cause degradation of proteins present in the
bacterial cell walls and slow down the bacterial growth, and as
a result have found variety of applications in the fields of
biodiagnostics, manufacture of optical fibres etc. [18].

The most remarkable mechanism of antibacterial activity of
silver nanoparticles was found to be the formation of free
radicals which take part in the oxidative damage of the cell
membranes of the bacteria [19,20]. Doping of silver ions onto a
titanium dioxide based surface resulted in the movement of the
absorption to a longer wavelength due to a change in the
electronic and optical properties of TiO2. Also TiO2 was a very
good supporting material for silver nanoparticles as it had a
small crystal size and high surface area.

Much of the work has been done on the efficacy of
nanoparticles in cleaning of water and environment and there is
very little literature on the effect of silver doped titanium
dioxide on pathogens from clinical samples.

Hence, this work was done to investigate the effectivity of
nanoparticles of TiO2 doped with 0.3% wt of silver prepared
by sol-gel technique, as an anti-bacterial agent against Pseudomonas aeruginosa from clinical samples.

Materials and Methods

This experimental study was conducted at Mallareddy Institute
of Medical sciences, Hyderabad over a period of 2 and half
years, between March 2014 and September 2016.

Initial preparation

A glass slide was cut into 3 equal parts with a glass cutter so
that 3 equal squares of the glass slides are formed. On this,
deposition of thin films of TiO2 doped with 0.3% wt of Ag was
done by sol-gel method (Courtesy Nano-Ram Technologies,
Bangalore). 3 Plain glass slides of the same size with no
nanoparticle coatings were taken as controls. All the glass
slides were properly labelled and covered with tissue paper and
then with silver foil individually. They were all then placed in
separate petri plates and sterilized in hot air over at 160˚C for 1h.

Collection of samples

Various clinical samples like urine, pus, sputum, swabs from
various sites, were inoculated with aseptic measures, onto
MacConkey agar and Blood Agar as per the regular protocol.
All these plates were labeled properly and incubated overnight
at 37˚C. Next day the growth was identified by the colony morphology on MacConkey and Blood agar and various
biochemical reactions and their antibiograms were put up on
Mueller Hinton Agar. The drug sensitivity pattern was
identified according to CLSI guidelines. 25 samples of Pseudomonas aeruginosa which were sensitive to all the drug
groups like ampicillin, cephalosporins, quinolones, etc. were
included into the study.

All gram positive organisms and all gram negative bacilli other
than P. aeruginosa were excluded from the study. Drug
resistant P. aeruginosa especially those resistant to
cephalosporins and metallo-beta lactam producers, were also
excluded from the study.

Processing of P. aeruginosa on TiO2 slide

One colony of P. aeruginosa was inoculated onto sterile
peptone water and incubated overnight at 37˚C. Next day the
peptone water was adjusted to 0.5 MacFarland’s standard to
make the working broth.

The 6 Petri plates, 3 in which TiO2 was coated and the 3 plain
slides were taken and the covering papers were removed. 100 l
was taken from the working broth and added onto all the slides
aseptically. 50 l was taken from this by microtitre pipette and
added onto sterile Mueller Hinton agar each and a well
inoculum was made. Primary, secondary, tertiary and tail
streaks were made from here and incubated at 37˚C overnight.

All the 6 slides were placed back in the respective plates. Of
the three nano particle coated and control slides, one plate each
was covered with a black paper and incubated at 37˚C in the
incubator. Of the other two plates, one was incubated at 37˚C
in visible light and the other in the BOD incubator at 37˚C with
the UV light on.

After 1 h, all the slides were taken and 50 l sterile distilled
water was added to each and mixed thoroughly. 50 l was taken
from this solution and added to another set of sterile MHA
plates, streaked and incubated at 37˚C overnight. The slides
were once again processed and incubated as above. This
process of adding the distilled water onto TiO2 coated and
washing was repeated at 2, 4, 6 and 8 h. 50 l from this was
taken each time and inoculated onto MHA plate and incubated
at 37˚C overnight.

The growth was observed the next day.

Results

There were 5 sets of plates from 6 slides-TiO2 coated slide and
control incubated in light, in dark and in UV light at 37˚C.

Out of them, the inoculum from the control slides showed
confluent growth even up to the tertiary and tail of the streak
lines, showing no reduction of the amount of viable bacteria
over time (Figure 1). Even after 8 h of treatment at light or
dark or under UV light, there was no reduction or difference in
the growth pattern of the organism although there was a
marginal reduction of growth in the plate which was incubated
in the UV light.

Figure 1: Control plates at different sources of light after 8 h of
treatment. a: after 8 h in dark; b: after 8 h in light; c: after 8 h in UV.

However, the reduction in the treatment of the organism with
TiO2 doped with 0.3% wt Ag, remarkable reduction in the
growth. After 3 h interval, growth was observed up to the
tertiary streak lines in the plates with organisms incubated in
the dark, however, there was slight growth in the well only
after 6 h of treatment Figure 2.

However, in the plates with nanoparticles incubated under
ultraviolet light, no growth of the organisms were seen in the 3rd hour itself, while around 10 colonies were seen after the
incubation of 2 h (Figure 4).

When the logarithmic scale of the organism was taken, it was
observed that the number of colony forming units/ml at the
start of the study in each condition of incubation was>106.
However, after one hour of incubation, it was observed that the
number of colonies reduced to one third (around 10000 cfu/ml)
when incubated under UV, while it remained almost constant
when incubated in dark. Within 2 h, the cfu/ml was reduced to
10 log while it took 6 h when incubated in light (Figure 5).
100% antibacterial effect was observed within 4 h when
incubated in UV, while it took over 6 h when incubated in
light, at which time there were double the number of organism
in dark as compared to those in light.

Figure 5: No of colonies on a logarithmic scale over time, incubated
in dark, light and UV.

Discussion

Implants made up of titanium dioxide have been highly used
for some time now in many practices in medicine, such as knee
implants, dental implants, screws for orthopaedic surgeries,
pacemakers etc.

Nanoparticles are said to exhibit very strong inhibitory effect
towards a large number of bacterial strains. Metal oxides
according to many studies are believed to be carrying a
positive charge while the cell walls of the microorganisms carry a negative charge. As a result, there is an electromagnetic
effect between these two structures, leading to oxidization and
ultimately the death of the organism [21].

Pseudomonas is associated with many hospital acquired
infections including biofilms formations in conditions such as
cystic fibrosis and in ventilators. Evaluation of effectivity of
nanoparticles on resistant bacteria such as Pseudomonas, Staphylococcus aureus etc., is becoming very difficult and
hence a reason for many studies [22].

The present study by treating with nanoparticles has shown a
marked reduction of Pseudomonas aeruginosa under all the
sources of light i.e. in dark, in visible light as well as under UV
illumination within 6 h of treatment. In fact with UV
illumination, there was no growth of viable bacteria within the
3rd hour of treatment itself, showing that the nanoparticles act
better at UV light rather than under visible light and in dark.

Although it is estimated that light is necessary for the
excitation of silver ions for antibacterial effect, it is observed
that even under dark, the efficacy of the nanoparticles is
maintained.

In a study by Sangchay et al. it was observed that E. coli was
eliminated much faster by TiO2 doped with Ag under UV
radiation [23]. More over as the exposure to UV radiation
increased, so did the efficacy of the nanoparticles.

ATCC strains of water pollutants such as E. coli and Klebsiella spp were tested against TiO2 doped with ZnO under UV
irradiation as well as under visible light. It was observed that
there was considerable reduction in the growth of the
microorganisms when incubated with TiO2 or in combination
with ZnO in UV or in visible light which corresponded to the
results of our study. This extent of the bacterial inhibition
depended on the concentration of the ZnO particles and also
the nature of the electromagnetic spectrum [24]. However in a
study with Cu as the dopant, there was no difference in the
number of viable counts of the fungal pathogens such as Candida with increase in the concentration of Cu, while there
was a considerable decrease in the bacterial counts [25].

In yet another study by Prasad et al. Ag and its various
concentrations on TiO2 showed high antibacterial effect against
the bacterial and fungal water pathogens. The results were in
accordance to our study on clinical pathogens.

The study of nanomedicine and its applications, although has
been known for a very long time, has taken enormous
proportions only recently. There has been a tremendous
amount of work in the field of water sterilization and
environmental cleaning. Silver ions have been used in dental
implants for a very long time now with spectacular success,
though only of later, the use of Ag in the form of nanoparticles
has spiked the interest of the researchers.

Our study has used the clinical samples which are capable of
causing infections in the human body at various sites and
observed the effect of TiO2 with Ag as an additive. This
present study has not been done on the metal implants in vivo,
which can be the topic of interest in further studies in future.

Conclusion

TiO2 with Ag as an additive is highly effective as an
antibacterial agent on many human pathogens including Pseudomonas aeruginosa, which is notorious in causing
hospital acquired infections. TiO2 with 0.3 wt (%) Ag is most
effective under UV irradiation and least in dark.

As today, most of the technology has become nano-oriented;
we should also modulate our treatment to incorporate the new
trends. Thus, more such tests on different surfaces and their
effects in the human body must be carried out in future so that
newer developments in science and medicine can be make our
quality of life better.